Analysis of mammalian cDNA and genomic clones for the catalytic subunit of the calmodulin (CaM)-dependent protein phosphatase, calcineurin (CN) indicate multiple molecular isoforms that result from alternative splicing of distinct genes. The evolutionary relationship between these genes has been characterized, as have their localizations on human chromosomes and differences in mRNA expression. To study the origins of this genetic complexity and its regulatory controls, we are comparing the corresponding genes in lower eukaryotes. In these simpler organisms primary structure is highly conserved in catalytic and regulatory domains, while regions of unrelated sequence near the carboxyl terminus suggest areas that may impart unique substrate specificity. In Neurospora intron/exon boundaries corresponded to those we have found in the mammalian gene, suggesting a step-wise evolution of DNA control features. In both Neurospora and Dictyostelium, mRNA expression appears to be developmentally controlled, and studies to examine promoter regulation are being pursued. A bacterially-expressed fungal catalytic subunit having high enzyme activity was produced and a set of deletion mutants of the mammalian protein delineated the minimum structural domain needed for CaM binding; a synthetic peptide based on this region exhibited an inhibitory constant of one-fourth 5 nM. Developmental studies on phosphodiesterase (PDE) showed a pattern of expression that coincided with synaptogenesis in most brain areas; the e.m. localization of PDE in neurons is now being studied to suggest its possible functional roles. A novel brain-specific CaM binding protein of 140 kDa (native MW) has been characterized that is localized primarily in limbic structures.